1. Field of the Invention
This invention is related to the field of probe based nucleic acid sequence detection, quantitation and analysis. More specifically, this invention relates to methods and kits, which prevent the generation of false positive results in probe based assays, particularly where said probes are fluorescently labeled PNA or LNA probes.
The invention is more specifically directed to methods and kits suitable for improving the specificity and/or reliability of diagnostic tests using probes. The methods and kits of this invention are particularly well-suited for fluorescence in situ hybridization.
2. Description of the Related Art
Nucleic acid hybridization is a fundamental physiochemical process, central to the understanding of molecular biology. Probe-based assays use hybridization for the detection, quantitation and analysis of nucleic acids. Nucleic acid probes have long been used to analyze samples from a variety of sources for the presence of nucleic acids, as well as to examine clinical conditions of interest in single cells and tissues. More recently, high affinity nucleic acid probe analogs and mimics have become the preferred reagents for hybridization assays.
Locked Nucleic Acid (LNA) and Peptide Nucleic Acid (PNA) are novel high affinity probes which provide higher sensitivity and specificity than conventional DNA probes. DNA is a biological material that plays a central role in the life of living species as the agent of genetic transmission and expression, whereas LNA and PNA are recently developed totally artificial molecules, conceived in the minds of chemists and made using synthetic organic chemistry. Although LNA and PNA can employ common nucleobases (A, C, G, T, and U) and can hybridize to nucleic acids with sequence specificity according to Watson-Crick base paring rules, they differ both structurally and functionally from DNA. Peptide Nucleic Acid, despite its name, is neither a peptide nor a nucleic acid, nor is it even an acid, but a non-naturally occurring polyamide backbone composed of (aminoethyl)-glycine subunits where the nucleobases are connected to the backbone by an additional methylene carbonyl moiety. (See: U.S. Pat. No. 5,539,082 and Egholm et al., Nature 365:566-568 (1993)). Due mostly to the fact that PNA carries a net neutral electrical charge, PNA can form hybrids extremely rapidly and stably with naturally occurring nucleic acids. LNA is a nucleic acid analog created by chemically joining the 2′ oxygen and 4′ carbon of a ribonucleoside through a methylene linkage. The highly rigid structure of the resultant locked 3′-endo conformation reduces the conformational flexibility of the ribose. The increased rigidity and local organization of the LNA phosphate backbone lowers the entropic penalty for hybridization of LNA probes as compared to DNAs of the same relative composition. These structural features provide PNA and LNA probes with higher affinity for target sequences and furthermore allow PNA and LNA probes to hybridize under conditions that are destabilizing to naturally occurring nucleic acids, such as low salt concentration or in the presence of guanidinium hydrochloride. These attributes enable PNA probes to access targets, such as highly structured rRNA and double stranded DNA, known to be inaccessible to DNA probes (See: Stefano & Hyidig-Nielsen, IBC Library Series Publication #948. International Business Communication, Southborough, Mass., pp. 19-37 (1997), (Fuchs, Appl Envir Micro 64 (12) 4973-82, 1998; Efficient poly(A)+ RNA selection using LNA oligo(T) capture. Technical note, Exiqon A/S, Denmark. LNA and PNA are useful candidates for investigation when developing novel probe-based hybridization assays because of their excellent hybridization features.
Probe based assays are useful in the detection, identification and quantitation of nucleic acids. Nucleic acid probes have long been used to analyze samples for the presence of nucleic acid from a bacteria, fungi, virus or other organism (See for example; U.S. Pat. Nos. 4,851,330, 5,288,611, 5,567,587, 5,601,984 and 5,612,183). Probe-based assays are also useful for examining genetically based clinical conditions of interest. Despite the high specificity of LNA and PNA probes as compared to naturally occurring nucleic acid probes, it is common to encounter sequence regions where it is very difficult to design a probe which allows exclusive detection of the desired target in a particular assay format.
The use of PNA probe mixtures have previously been described as a way to target two adjacent target sequences (US2002058278) as a means to increase specificity, however, that concept required each of the two PNA probes hybridizing the adjacent target sequences to be extended with an arm segment capable of forming a triplex with a third labeled PNA probe, such that a total of three probes were required. In addition, the use of a fourth probe or antibody was proposed.
Blocker probes are nucleic acid or non-nucleic acid probes that can be used to suppress the binding of the probing nucleobase sequence of the probe to an unwanted target sequence. Preferred blocker probes are PNA probes (See: Coull et al., U.S. Pat. No. 6,110,676). Typically blocker probes are closely related to the probing nucleobase sequence and preferably they comprise one or more single point mutations as compared with the probe sought to be detected in the assay. Blocker probes operate by hybridizing to non-target sequences, thereby preventing these sequences from being available for hybridization with the detectable probe. The competition set up between the blocker probe and the detectable probe ensures that each probe will only bind to highly complementary targets, increasing the specificity of the assay. Generally and preferably, blocker probes are directed towards a particular single non-target sequence which differs by at least one nucleobase from the target sequence.
The “Hybridization Probes” method (U.S. Pat. No. 6,174,670) describes use of two DNA probes which hybridize to adjacent target sequences, where one DNA probe is labeled with a fluorophore (donor) and the other DNA probe is labeled with another fluorophore (acceptor), such that simultaneous hybridization of the two probes facilitate Fluorescence Resonance Energy Transfer (FRET). The method teaches that FRET occurs as the energy from the excitation of the donor fluorophore is transferred to the donor fluorophore where it is emitted at the emission wavelength of the donor fluorophore. The combined specificity of the two probes is greater than that of either probe alone, as the detectable signal is dependent on the specific hybridization of two DNA probes. Use of Hybridization Probes is limited to selected donor-acceptor fluorophore pairs and instrumentation, such as the LIGHTCYCLER™, with a special combination of excitation filter for the donor fluorophore and emission filter for the acceptor fluorophore. This method is not directly applicable in, for example, fluorescence in situ hybridization assays using standard fluorescence microscope filter sets.
It would be desirable to have a method for improving the specificity for a target sequence that employs quenching of the fluorescence from binding of fluorophore-labeled probes to unwanted target sequences.